The tryptophan (trp) operon of E. coli consists of 5 contiguous structural genes and their associated control elements. Existing procedures permit isolation of selected regions of trp messenger RNA labelled with 32P (in vivo or in vitro), by sequential hybridizations to trp-transducing phage DNA. Experiments utilizing current techniques of mRNA labelling, isolation, and sequencing are proposed in two main areas: (1) Primary structure of regulatory and punctuation regions in trp mRNA. These projects continue and complement my present work, and include nucleotide sequence determination of (a) the ribosome-binding sites for trpD, trpC, and trpB, (b) the intercistronic regions between trpE-trpD, trpD-trpC, and trpC-trpB, and (c) the 3' region of trp mRNA beyond trpA. Some of these sequences occur outside structural gene mRNA and probably serve regulatory functions, since they are not translated. They will be compared with others of similar types in E. coli and elsewhere. This is expected to yield some understanding of the mechanisms involved in controlling translation and transcription within a bacterial polycistronic operon. (2) Regulatory role of the trp "leader" region. Sequences in this region are involved in regulation of operon expression, but the precise mechanisms are unknown. A ribosome-binding site near the 5' end of the leader mRNA suggests the existence of a new gene in the trp operon. To test this prediction, isolation of a gene product will be attempted, in studies of trp-specific polypeptide synthesis in vivo and in vitro. Leader mRNA will also be tested for its susceptibility to ribonucleases that are involved in "processing" functions, to determine whether this region of mRNA is processed or possesses detectable secondary structure.